Method for the extraction of components made from organic material

ABSTRACT

The present invention relates to a process for extracting constituents from organic material, comprising the step of extracting with a monophasic mixture of 50% by volume to 90% by volume of methanol and 50 to 10% by volume of water and optionally 0% by volume to 40% by volume of a further solvent or solvent mixture.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/494,419, which is a 35U.S.C. 371 National stage filing ofInternational Application No. PCT/EP2002/12551, filed Nov. 11, 2002,which claims priority to German Application No. 10155517.2-43, filedNov. 13, 2001 and German Application No. 10203551.2, filed Jan. 29,2002. The entire contents of each of these applications are herebyincorporated by reference herein in their entirety.

FIELD OF INVENTION

The present invention relates to a process for extracting constituentsfrom organic material, comprising the step extracting with a monophasicmixture of 50% by volume to 90% by volume of methanol and 50 to 10% byvolume of water and, optionally, 0% by volume to 40% by volume of afurther solvent or solvent mixture.

DESCRIPTION OF THE BACKGROUND

The understanding of the biochemical synthetic pathways in themetabolism of animal or plant cells, including microorganisms such asbacteria, fungi and algae, or mammalian cells, remains very rudimentary,even though the main synthetic pathways are known. To date, thedetermination of physiological states during growth, development or as aresponse to environmental stress is essentially limited to the study ofindividual target molecules such as, for example, RNA and proteins.However, changes in the mRNA or protein level or their activity canfrequently not be correlated with changes in the metabolism or indeedwith phenotypic functions.

Cellular constituents or metabolites are frequently analyzed directlyeither by specific enzymatic reactions, immunoassays or on the basis ofchromatographic methods, which identify certain substances by theirretention times or coelution with reference substances. As described inKatona, J. Chromatography 1999, 847, 91-102, most of the prior art onlydeals with the analysis of few, specific components, for example acidsor sugars. Most of the known processes limit a comprehensive biochemicalanalysis by the following facts: (a) they are not open, i.e. it is onlypossible to obtain information on metabolites which are already known,(b) they are very labor-intensive since they are frequently based onindividual assays; (c) their resolution is only very poor, and theresult is therefore a profile which only has a very low degree ofcomplexity and contains little information, (d) they only covermetabolites in a cell in only one status, for example a growth phase orunder the effect of a stress factor, and dynamic changes in the cell cantherefore not be monitored, or (e) they only cover few of the classes ofsubstances present in the organism, for example only sugars or onlyfatty acids or only certain molecular weights, but not the broad rangeof polar or unpolar substances. There exist only first attempts todemonstrate that metabolites not only constitute intermediate or endproducts, but also act as sensors and regulators.

The comprehensive and quantitative determination of the metabolites andconstituents present in an organic sample (independently of whether thisdetermination is limited to various classes of substances, givendevelopmental stages or types of material, i.e. independently of whetherit takes the form of metabolic fingerprinting, metabolic profiling ormetabolomics) enables the direct study of immediate effects of growth,development or stress on the entire organism or parts thereof and isthus helpful as essential part of functional genome analysis in thedetermination of gene functions. Processes for analyzing metabolicprofiles, in particular when they are suitable for analyzing substantialnumbers of samples, permit the study of the complexity of the regulatoryinteractions at all levels and all stages and, last but not least, theassessment of the safety and value of genetically modified organisms.

The most advanced aspect is the determination of complex metabolicprofiles in diagnostic screens, but first profiles have also recentlybeen described for plants (for a review see Trethewey, Curr. Opin.Plant. Biol. 1999, 2, 83-85). Thus, Sauter (ACS Symposium Series 1991,443 (Synth. Chem. Agrochem. 2), American Chemical Society, Washington,D.C., 288-299) demonstrates the modification of constituents in barleyfollowing treatment with various herbicides. Sauter describes theweighing of frozen plant samples and extraction thereof with 100%ethanol as advantageous method. Following filtration, drying andsilylation, the samples can be separated via capillary columns. Between100 and 200 signals were detected and identified with the aid ofreference substances via their retention coefficients in gaschromatography (GC) or via gas chromatography/mass spectrometry analysis(GC/MS).

Fiehn, Nature Biotechnology 2000, 18, 1157-1161 describes thequantification of 326 substances in Arabidopsis thaliana leaf extracts.To compare four different genotypes, frozen plant samples werehomogenized in a complicated procedure, extracted with 97% methanol andbrief heating, and, after addition of chloroform and water, a multi-stepprocedure gave a polar and an unpolar phase which were then analyzed byLC/MS and GC/MS (see also Fiehn, Anal. Chem. 2000, 72, 3573-3580;http://www.mpimp-golm.mpg.de/fiehn/blatt-protokoll-e.html). Following avery similar method, Roessner, The Plant Journal 2000, 23, 131-142,extracts plant constituents with methanol and compares the profiles ofpolar metabolites of in-vitro potato plants and potato plants grown insoil.

Gilmour, Plant Physiology 2000, 124, 1854-1865 extracts sugar fromlyophilized Arabidopsis leaves in 80% ethanol following incubation for15 minutes at 80° C. and incubation overnight at 4° C. Strand, PlantPhysiology 1999, 119, 1387-1397 extracts soluble sugars and starch twicein succession, likewise at 80° C. and for 30 minutes and in 80% ethanolwith Hepes, pH 7.5. The material is then reextracted twice at this hightemperature to improve the result of the extraction, once with 50%ethanol/Hepes, pH 7.5, and once with Hepes, pH 7.5.

These methods described in the prior art only permit limited automationwhich, moreover, can only be realized in the form of a complexprocedure. In particular the processing of large sample numbers, thedetermination of the effect of a variety of stress factors on themetabolism of the organisms or the observation of dynamic processes,which requires a continuous analysis of samples during windows which areoften very short, require processes

-   (a) which are rapid, i.e. for example that fixing and analysis of    the samples is effected within a short period after sampling,-   (b) which are highly reproducible, i.e. for example that an analysis    carried out with a large number of different samples gives results    within a very narrow error margin,-   (c) which are simple to handle, i.e. for example that the process    can be automated and does not require complex or laborious    procedures,-   (d) which are open, i.e. for example that a large number of    substances can be analyzed, and/or-   (e) which are sensitive, i.e. for example that the analysis    identifies even small changes in substance concentrations and small    amounts of substance.

Many processes have the disadvantage that they are only suitable for theanalysis of small sample numbers. With a larger number of samples, it isnot possible to ensure sample stability, and thus the reproducibility ofthe results. A comprehensive continuous analysis of biological material,for example animal samples or plant samples, or for example theinteraction between a substance, or substances, and organisms in complexsystems and their course over time is thus not possible.

SUMMARY OF THE INVENTION

The present invention overcomes the problems and disadvantagesassociated with current strategies and designs and provides newprocesses for extraction constituents from organic material.

One embodiment of the invention is directed to processes for extractingconstituents from organic material, comprising the steps of extractingwith a monophasic mixture of 50% by volume to 90% by volume of methanoland 50% by volume to 10% by volume of water and, optionally, 0% byvolume to 40% by volume of a further solvent or solvent mixture; andextracting with a monophasic, unpolar solvent or solvent mixture,wherein the combined extracts form one phase. Another embodiment of theinvention is directed to constituents extracted by a process of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

It is an object of the present invention to find a process which permitsovercoming the abovementioned shortcomings of the prior-art processesand which makes possible the rapid and simple analysis of, in particulara substantial number of, samples with a high degree of sensitivity andreproducibility.

We have found that this object is achieved by the embodimentscharacterized in the claims of the present invention.

Accordingly, the application relates to a process for extractingconstituents from organic material, comprising the step (a) extractingwith a monophasic mixture of 50% by volume to 90% by volume of methanoland 50% by volume to 10% by volume of water. In accordance with theinvention, 0% by volume to 40% by volume of the mixture optionallyconsist of a further solvent or solvent mixture, the total mixtureforming one phase. Further solvents or solvent mixtures are listedhereinbelow.

The term “organic material” refers to any organic or biologicalmaterial, such as material of plants, animals, microorganisms, forexample protists, fungi, bacteria, algae, viruses and the like, such asorganisms separated from culture material, body fluids such as blood,lymph, secretions, or foodstuffs, feedstuffs and other animal or plantproducts. Likewise, it refers to culture material in which organismslive, i.e. for example also after the organisms have been removed, forexample media for growing microorganisms such as protists, for examplekinetoplasts, plasmodia or bacteria, for example Gram-positive orGram-negative bacteria, or algae or fungae, for example yeasts, oranimal or plant cells.

The term “extraction” or “to extract” as used in the present inventionrefers to the transfer of substances contained in a solid or liquidsample, for example constituents of organic material, into therespective solvent or solvent mixture using unpolar to polar solvents orsolvent mixtures. The hydrophilic constituents, including, for example,metabolites, dissolve in a polar solvent, such as, for example, water,while the hydrophobic constituents, including, for example, metabolites,of the material are dissolved in a lipophilic solvent.

“Polar solvents or solvent mixtures” refers to a solvent or solventmixture with a polarity index of from 4 to 10.2, preferably from 5 to 7,more preferably from 5.5 to 6.5, as described by Kellner, AnalyticalChemistry, Weinheim, 1998, p. 195. Polar solvents are, for example,water including aqueous solutions, or polar aprotic or protic organicsolvents, for example alkyl alcohols with an alkyl radical having 1 to 6carbon atoms, for example methanol, ethanol, 1-propanol, 2-propanol,butanol, pentanol, hexanol or, for example, acetone, acetonitrile, ethylacetate, dimethyl sulfoxide or N,N-dimethylformamide, or other solventswith a polarity greater than or equal to 0.50, as stated for example inKüster/Thiel, Rechentafeln für die Chemische Analytik [Nomograms fortechnical analysis], Walter de Gruyter, Berlin/N.Y. 1993, p. 359, or aremixtures of these. For example, a solvent mixture of 80% methanol/20%water used in accordance with the invention has the polarity index of6.1 as defined by Kellner, 1998.

In the following text, “unpolar solvents or solvent mixtures” refers tosolvents or solvent mixtures which have a polarity index as defined byKellner, Analytical Chemistry, Weinheim, 1998, p. 195, which falls shortof the polarity index of the extractant of the polar phase by 0.3 ormore. More preferably, the polarity index as defined by Kellner, 1998,falls short of that of the extractant of the polar phase by 0.5, evenmore preferably by 1, most preferably by more than 2. Accordingly, thepolarity index of the unpolar solvent is especially preferably 5.5 to 1,more preferably 5 to 2, most preferably 4.5 to 3.5 as defined byKellner, 1998. Thus, for example, a solvent mixture of 40% methanol/60%dichloromethane employed in accordance with the invention has a polarityindex of 3.9 as defined by Kellner, 1998. “Unpolar solvents” alsoinclude, for example, organic solvents, for example halogenated solventssuch as chloroform, dichlorornethane, carbon tetrachloride or aliphaticsolvents such as hexane, cyclohexane, pentane, heptane and the like, oraromatic solvents such as, for example, toluene or benzene, or etherssuch as for example, tert-butyl methyl ether, diethyl ether ortetrahydrofuran, or other solvents with a polarity of less than 0.50 asstated for example in Küster/Thiel 1993, or mixtures of these.

A “polar phase” or a “polar extract” refers to a phase or an extractwhich is obtained by extraction with a solvent or solvent mixture with apolarity index of from 4 to 10.2, preferably from 5 to 7, morepreferably from 5.5 to 6.5 as defined by Kellner, Analytical Chemistry,Weinheim, 1998, p. 195, or which is obtained by extraction with a polarsolvent or solvent mixture as stated above.

An “unpolar phase” or an “unpolar extract” refers to a phase or extractwhich has a lower polarity or a lower polarity index relative to thepolar phase or the polar extract and with which substances of mediumpolarity to unpolar substances can be extracted better, such as, forexample, in the case of extraction with unpolar solvents or solventmixtures as stated above. In accordance with the invention, an “unpolarphase” or an “unpolar extract” is obtained by extraction with a solventor solvent mixture with a polarity index as defined by Kellner,Analytical Chemistry, Weinheim, 1998, p. 195, which falls short of thepolarity index of the solvent or solvent mixture of the polar phase orof the polar extract by 0.3 or more. More preferably, the polarity indexfalls short of the polarity index of the solvent or solvent mixture ofthe polar phase by 0.5, even more preferably by 1 most preferably bymore than 2, as defined by Kellner, 1998.

The term “constituents” refers to polar and unpolar compounds, forexample “metabolites”, which originate from the catabolic or anabolicreactions of the metabolism or which are taken up by organisms fromtheir environment. This relates to compounds which are localizedcellularly or, in more complex organisms, also extracellularly, forexample in body fluids. In the culture of microorganisms or otherorganisms, the constituents of these cultures, for example of theculture medium, are also encompassed. The concentration of a constituentis affected by external factors (environmental conditions, nutrientconditions, stress situation) or by internal conditions (development,regulations, changes owing to genetic influence) which the organisms aresubjected to. The term refers both to what are known as primarymetabolites and to secondary metabolites. “Primary metabolites” refers,as a rule, to those metabolites which are products of catabolic andanabolic pathways and which are of fundamental importance for the celland thus more or less identical for all cells. “Secondary metabolites”refers, as a rule, to compounds which are formed predominantly insecondary pathways, for example in the case of stress situations, suchas hunger or deficiency conditions, or after the active growth phase ofthe cell has ended and for which no discernible cellular function isknown in many cases (see also Römpp Lexikon Biotechnologie, New York,1992). Constituents are therefore understood as meaning for examplepolar and unpolar substances such as carbohydrates, amines (inparticular amino acids), tetrapyrroles, lipids, steroids, nucleotides,nucleosides, cofactors, coenzymes, vitamins, antibiotics, hormones,peptides, terpenes, alkaloids, carotenoids, xanthophylls, flavoids andthe like, and the substances of the respective metabolic pathways,without the above or the following enumeration in any form beingconsidered as limiting.

Carbohydrates encompass for example the carbohydrates of thecarbohydrate metabolism, for example glycolysis, gluconeogenesis, forexample trioses, tetroses, pentoses, for example furanoses, or hexoses,for example pyranoses or heptoses, of the polysaccharide metabolism, ofthe pyruvate metabolism, of the acetyl coenzyme A metabolism, di- oroligosaccharides, glycosides, hexose derivatives, deoxy hexoses,carbohydrates of the pentose metabolism, of the amino sugar metabolism,of the tricarboxylic acid cycle of the glyoxylate metabolism and thelike, or other substances of the respective metabolic pathways.

Amino acids encompass for example the amino acids of the amino acidsmetabolisms such as, for example, in the ammonia metabolism, or of thesulfur metabolism, the urea cycle, or their derivatives, for examplearomatic or nonaromatic amino acids, polar uncharged, unpolar,aliphatic, aromatic, positively-charged, negatively-charged amino acids,branched-chain or straight-chain, essential or nonessential amino acids,or other substances of the respective metabolic pathways.

Tetrapyrroles encompass, for example, substances of the protoporphyrinmetabolism, the haemoglobin metabolism, the myoglobin metabolism, thevarious cytochrome metabolisms, the photosynthetic metabolisms and thelike, or other substances of the respective metabolic pathways.

Lipids encompass for example saturated or unsaturated, essential ornonessential fatty acids, acyl-CoA compounds, triacylglycerides, lipidsof lipogenesis or lipolysis, phospholipids, for exampleglycerophospholipids, ether lipids, sphingophospholipids, glycolipids,or the substances of the respective metabolic pathways.

Hormones encompass steroids or nonsteroid hormones, for example peptidehormones or, for example, eicosanoids or octadecanoids.

Steroids encompass for example the substances of the cholesterolmetabolism, hopanoids, plant steroids such as phyto- and mycosterols,insect hormones, isoprenoids, steroid hormones, gestagens, androgens,oestrogens, corticosteroids or the substances of the respectivemetabolic pathways.

Nucleotides and nucleosides encompass, for example,deoxyribonucleotides/nucleosides and ribonucleotides/nucleosides, their5′-phosphate derivatives, purines, pyrimidines or their derivatives, forexample cyclized, methylated and/or acetylated nucleoside or nucleotidederivatives and the like, or other substances of the respectivemetabolic pathways.

Likewise included are substances which play a role in these metabolicpathways. “Other substances of the respective metabolic pathways” refersto the respective intermediates in the biosynthesis, in the conversion,the transport or the metabolism of the substances mentioned. An overviewof many metabolites can be found for example in Michal, BiochemicalPathways, Berlin, 1999 or in KEGG, Kyoto Encyclopedia of Genes andGenomes, Institute of Chemical Research at Kyoto University, Japan (z.B.http://www.genome.adjp/dbget/ligand.html), which are expresslyincorporated herewith.

The term “water” refers to any type of aqueous solution, including, forexample, deionized, demineralized, distilled or twice-distilled water.One or more substances which preferably improve the extraction,stability or solubility of the constituents of the organic material orwhich bring about preferred properties, for example pH value,conductivity, salt concentration and the like, may also be dissolved inthe water or mixed therewith, as is the case, for example, in saltsolutions or buffer solutions.

In the prior art, polar substances are, as a rule, extracted with purealkyl alcohols such as ethanol (Sauter, 1991, Strand, 1999, Gilmour,2000) or methanol (Fiehn, Anal. Chem. 2000 and Nature Biotechnology2000, Roessner, 2000), alkyl alcohols which are mixed with water orbuffer solutions, or with water or buffer solutions.

Water has very good extractive properties for polar substances. However,the fact that cellular processes, which, as a rule, have previously beenstopped by freezing or freeze-drying, are reactivated in aqueoussolutions is a disadvantage. This can bring about the enzymaticdegradation or conversion of various metabolites and leads to a changein, and thus falsification of the concentrations or ratios within theseextracts. As a rule, one attempts to prevent these undesired sidereactions by working on ice. However, this has considerabledisadvantages, both for the extraction efficacy and for working up largenumbers of samples. Losses in sensitivity and a deviation of the actualcellular state at the point in time of harvesting are unavoidable.

Ethanol or methanol are used firstly because they have polar propertiesand thus extract hydrophilic constituents to a sufficient degree, and,secondly, since after their addition to a cell extract the activity ofthe cell extracts is inhibited owing to the toxic, denaturing effect ofthe alcohol. Thus, further reaction of the metabolites can no longertake place, and the cells are “frozen” at the metabolic stage ofprecisely this moment, However, the disadvantage is that some polarclasses of metabolites are only sparingly soluble in methanol orethanol. This leads to a loss in sensitivity and may also have an effecton the reproducibility of the results.

To overcome the disadvantages of an extraction with pure alcohol or purewater Strand, 1999 and Gilmour, 2000, amongst others, use ethanol/watermixtures for the extraction of sugars and incubate the starting materialfor 15 minutes or 30 minutes respectively, at 80° C. in this mixture.This is followed in each case by further complicated extraction steps inorder to improve the insufficient extraction efficacy: Strand, 1999,establishes an extraction cascade of, thus, in total fourhigh-temperature steps: first twice with 80% ethanol/20% Hepes, then 50%ethanol/50% Hepes and finally 100% Hepes, in each case 30 minutes at 80°C., while Gilmour, 2000, incubates overnight at 4° C. after thehigh-temperature step. An extraction in four steps, or additionallyovernight after the high-temperature step, however, is very timeconsuming and labor-intensive. The attempt to compensate for theextraction properties of ethanol, which are poorer than those of water,by high temperatures and long extraction times can additionally lead todecomposition of the constituents and thus reduces the number ofsubstances to be detected and the sensitivity of the system.

It has now been found that the monophasic mixture according to theinvention of 50% by volume to 90% by volume of methanol and 10% byvolume up to 50% by volume of water leads to a very good extractionyield. The process according to the invention leads to a higher yieldthan is the case with an extraction in pure methanol or ethanol.Moreover, the stability of the extract is increased over that of a pureextraction in water, and the reproducibility of the process is thusimproved substantially. In contrast to the extraction with ethanol/watermixtures, the yield is sufficiently high for a single extraction step tosuffice for isolating a very large number of constituents. In processesbased on the extraction step according to the invention for isolatingpolar substances from plant cells, the number of analytes was onlylimited by the analytical method. A very high reproducibility wasachieved.

The mixture preferably comprises at least 70% by volume of methanol,with at least 75% by volume being more preferred, 90% by volume likewisebeing preferred, 85% by volume being more preferred, and 80% by volumeof methanol being most preferred.

Likewise preferred are 10% by volume to 50% by volume of water, with 15%by volume to 40% by volume of water being more preferred and 30% byvolume of water being even more preferred. 20% by volume of water aremost preferred. The process according to the invention is thus mostpreferably carried out with a mixture of 80% by volume of methanol and20% by volume of water. If appropriate, the mixture may also comprisesmall amounts of another solvent or solvent mixture, for exampledichloromethane, but less than 10% by volume are preferred and less than5% by volume are more preferred; most preferably, no other solvent ispresent in the mixture.

This first extraction step for polar substances can be followed by oneor more further extraction step(s) (a′) with a solution with a higherwater content or only with water or with a different alcohol, forexample an alkyl alcohol with an alkyl radical having 1 to 6 carbonatoms, for example methanol, ethanol, 1-propanol, 2-propanol, butanol,pentanol, hexanol, or, for example, acetone or acetonitrile, ifappropriate as a mixture with water or a mixture of differentcomponents. Methanol or a methanol/water mixture is most preferred. Ifappropriate, step (a) or (a′) may be repeated once or more than once.

In an especially preferred embodiment, only a single extractionfollowing step (a) is carried out.

Moreover, the process according to the invention may comprise step (b):extracting with a monophasic unpolar solvent or solvent mixture.

The solvent or solvent mixture in step (b) is more unpolar than thesolvent or solvent mixture in step (a). According to the invention, thepolarity index (as defined by Kellner, 1998) of the solvent or solventmixture in step (b) falls short by 0.3 or more of that of the extractantof the polar phase in step (a). More preferably, the polarity index asdefined by Kellner, 1998 falls short by 0.5, more preferably by 1, mostpreferably by more than 2, than that of the extractant of the polarphase. Preferably, the polarity index as defined by Kellner, 1998, ofthe solvent in (b) is from 5.5 to 1, more preferably 5 to 2, mostpreferably lower than 4.5 to 3.5. A solvent mixture employed inaccordance with the invention of 40% methanol/60% dichloromethane thus,for example, has a polarity index of 3.9 as defined by Kellner, 1998.

Unpolar solvents or solvent mixtures have been described above, forexample organic polar solvents or a mixture of one or more polarsolvents such as, for example, alkyl alcohols having an alkyl radicalwith 1 to 6 carbon atoms, for example methanol, ethanol, 1-propanol,2-propanol, butanol, pentanol, hexanol or, for example, acetone,acetonitrile, ethyl acetate, dimethyl sulfoxide orN,N-dimethylformamide, or other polar solvents with a polarity ofgreater than or equal to 0.5, for example as stated in Küster/Thiel1993, and one or more unpolar organic solvents described above, forexample halogenated solvents such as chloroform, dichloromethane, carbontetrachloride, or aliphatic solvents such as hexane, cyclohexane,pentane, heptane and the like, or aromatic solvents such as, forexample, toluene or benzene, or ethers such as, for example, tert-butylmethyl ether, diethyl ether or tetrahydrofuran, or other solvents with apolarity of less than 0.5 as stated in, for example, Küster/Thiel 1993.

A solvent component which is preferably employed in step (b) is onewhich is immiscible with water so that, upon a subsequent phasecombination, phase separation into an unpolar phase and a polar phasecan subsequently be brought about, However, the component is preferablymiscible with methanol. A low boiling point, for example of 100° C. orless, more preferably 80° C., even more preferably 60° C. and mostpreferably 40° C. or less under atmospheric pressure is furthermoreadvantageous since removal of the solvent or solvent mixture can then beeffected more rapidly and at lower temperatures under milder conditionsfor the constituents.

Halogenated solvents are preferred as solvents in step (b). Especiallypreferred as a mixture of methanol or ethanol and chloroform, pentane,hexane, heptane, cyclohexane, carbon tetrachloride or dichloromethane. Amixture of methanol or ethanol with chloroform or dichloromethane ismore preferred. The mixture in step (b) is preferably composed of 30% byvolume to 60% by volume of methanol or ethanol, a mixture with 40 to 50%by volume of methanol or ethanol being preferred. The use of methanol ismost preferred. Preferably, the dichloromethane or chloroform amounts to40 to 70% by volume, the use of dichloromethane being preferred. Theremaining 0% by volume to 30% by volume can optionally be composed of a“further solvent or solvent mixture” which form one phase with theabovementioned mixture, for example ethanol or chloroform. It ispreferred that no “further solvent or solvent mixture” is present.

If appropriate, the mixture in step (b) may also comprise small amountsof water, preferably 0% by volume to 20% by volume, with 10% by volumebeing more preferred, 5% by volume or less being even more preferred; nowater in the mixture is most preferred.

In a preferred embodiment, the process according to the invention thuscomprises the following step (b): extracting with a mixture of 30% byvolume to 60% by volume of C₁- to C₆-alkyl alcohol, acetone,acetonitrile, ethyl acetate, dimethyl sulfoxide orN,N,-dimethylformamide and 40% by volume to 70% by volume of chloroform,dichloromethane, pentane, hexane, heptane, cyclohexane or carbontetrachloride.

Surprisingly, it has been found that particularly good extraction ispossible with a specific ratio of methanol and dichloromethane. Step (b)of the process according to the invention is therefore most preferablycarried out with a mixture of 30 to 40% by volume of methanol and 60 to70% by volume of dichloromethane. 40% by volume of methanol and 60% byvolume of dichloromethane are most preferred.

If appropriate, step (b) can be repeated once or more than once and/orone or more further optional step(s) (b′) with a higher content ofaprotic solvent or pure aprotic solvent can follow.

In an especially preferred embodiment, step (b) is carried out onlyonce.

The steps of the process according to the invention, in particular steps(a) and (b), can be carried out in parallel, for example the sample isfirst divided and the two extracts are then prepared separately, or insuccession, for example when, after the 1^(st) extractant has beenremoved, the same sample is treated with a 2^(nd) extractant, with thesequence (a)(b) being just as possible as the sequence (b)(a), andintermediate steps also being possible. It is also possible to combinethe steps with other steps. The sequence (a)(b) is most preferred.

Thus, the process according to the invention, which comprises steps (a)and (b) and, optionally, also steps (a′) and (b′), allows the analysisof a large number of samples for polar and unpolar metabolites to becarried out in an automated fashion, rapidly and with a high degree ofreproducibility.

In a further step, the extracts from step (a) and step (b) are, in theprocess according to the invention, combined post-extraction to give aphase mixture. Mixing of the two extracts results, as a rule, in theformation of one or two phases, for example a polar phase and an unpolarphase.

It is advantageous to treat the combined mixture of (a) and (b) with oneor more analytical standards, for example internal standards and/orchromatographic standards. Examples of such standards can be compoundswhich do not occur in the natural samples, but which are similar to thesubstances analyzed, including isotope-labeled, radiolabeled orfluorescence-labeled substances, as in the case of sugars for exampleribitol or alpha-methylglucopyranoside, in the case of amino acids forexample L-glycine-2,2-d₂ or L-alanine-2,3,3,3-d₄, in the case of fattyacids or their derivatives in particular odd-numbered fatty acids ortheir methyl esters, for example methyl undecanoate, methyl tridecanoateor methyl nonacosanoate. The standards may also be added individually tothe respective extract of step (a) or (b), independently of whether theextracts are combined or not.

In one embodiment, solvents or solvent mixtures which additionallycomprise up to 5% by weight, more preferably up to 3% by weight and evenmore preferably up to 1% by weight, of buffer salts, acids and/or basesare used for extraction in the process according to the invention.Volatile buffer systems are preferred. Thus, substances which can beemployed in accordance with the invention are, for example, ammoniumformate solution, ammonium carbonate solution, ammonium chloridesolution, ammonium acetate solution or ammonium hydrogen carbonatesolution and/or an acid, for example formic acid, acetic acid,trifluoroacetic acid, pentafluoropropanoic acid, heptafluorobutanoicacid, nonafluoropentanioic acid, undecafluorohexanoic acid,tridecafluoroheptanoic acid or pentadecafluorooctanoic acid and/or abase such as, for example, triethylamine, pyridine or ammonia.

It is particularly advantageous when, in the extraction processaccording to the invention, the combined extracts form a phase to whichstandards can then preferably be added. The advantage of the repeatedmonophasic mixing of the two extracts prior to phase separation is thatresidues of polar substances from the unpolar extraction migrate intothe polar phase and, vice versa, residues of unpolar compounds migratefrom the polar phase into the corresponding unpolar phase.

Such a monophasic mixture is obtained, for example, when the extractionin step (a) is carried out with 80% by volume of methanol and 20% byvolume of water and in step (b) with 40% by volume of methanol and 60%by volume of dichloromethane and the two extracts are then combined witheach other. Thus, all of the constituents and, optionally for examplethe standards, are advantageously located in one phase.

The two phases can be separated by processes known to the skilledworker, see, for example, Bligh and Dyer, Can. J. Biochem. Physiol.1959, 37, 9111-917, for example by addition of an unpolar, in particularan organic (for example dichloromethane), or a polar solvent or solventmixture, in particular an aqueous solution, for example a buffer, or byaddition of both an unpolar and a polar solvent or solvent mixture ashave been described above. This gives rise to a phase comprising thepolar constituents (“polar phase”/“polar extract”) and a phasecomprising the constituents of medium polarity to unpolar constituents(“unpolar phase”/“unpolar extract”). Phase separation is preferablyachieved by addition of the solvents used for the extraction, inparticular by addition of methanol, dichloromethane and/or water. Aftercombining, the phases are separated again and can be derivatized and/oranalyzed, depending on their further use.

Instead of, or after, a phase separation, fractionation into two or morefractions can also be carried out, in the process according to theinvention, by means of solid-phase extraction. Fractionation into aplurality of fractions has the advantage that the derivatization methodsand analytic methods can be adapted better to suit the respective groupsof substances. Thus, in particular fractions which predominantlycomprise triglycerides are transesterified prior to analysis, forexample to give methyl esters. Solid-phase extraction is particularlysuitable for automation.

In a further embodiment, the process according to the invention thuscomprises one or more steps for derivatizing, chromatographing and/oranalyzing the constituents, for example from the extracts obtained, orthe phases. The extracts or the phases are preferably derivatized,chromatographed and analyzed in the following steps of the processaccording to the invention. To analyze the extracts further, certainconstituents must be derivatized, depending on the separation andanalytical methods used. Thus, derivatization is preferred forgas-chromatographic separation (GC), while derivatization is, as a rule,not necessary for separation by liquid chromatography (LC). Ifappropriate, an analytical method which does not involve chromatographicseparation is also possible for example mass spectrometry (MS), atomicabsorption spectrometry (AAS) or nuclear resonance spectrometry (NMR).

In a further preferred embodiment, the extraction process according tothe invention additionally comprises at least one of the followingfurther steps:

-   i) freezing the material, preferably rapid freezing of the material    obtained, for example the harvested material;-   ii) freeze-drying of the material;-   iii) homogenizing and dispersing the material;-   iv) evaporating an extract or a phase to dryness, in particular the    polar and the unpolar phase, in particular following phase    separation and/or transesterification/esterification in the phase;-   v) carrying out an esterification/transesterification in the unpolar    phase;-   vi) carrying out an oxime formation in the unpolar phase;-   vii) carrying out an oxime formation in the polar phase;-   viii) carrying out a trialkylsilylation in the unpolar phase; and/or-   ix) carrying out a trialkylsilylation in the polar phase.

The process according to the invention advantageously contains theindividual abovementioned steps, more preferably more than one of theabovementioned steps, and most preferably all of the abovementionedsteps, with the approximate sequence stated hereinabove or hereinbelowbeing especially preferred.

In accordance with the invention, it should be avoided that enzymaticprocesses which alter the constituent spectrum take place in the samplesup to extraction.

After harvesting, the material is therefore advantageously cooledinstantly, better frozen instantly, to prevent any enzymatic activity inthe sample or in the material in this manner and thus to avoid analtered distribution of the constituents. Preferably, freezing of thematerial after obtaining or harvesting it is carried out in less than 60s, with 30 s being more preferred and 15 s or less being most preferred.If the material takes the form of plant material, sampling can beeffected directly in the phytotrone chambers. After obtaining thematerial, the latter is advantageously weighed rapidly and then frozenrapidly at a low temperature, for example in liquid nitrogen, and storedfor example at −80° C. or in liquid nitrogen.

Freeze-drying of the material is also advantageous. Freeze-dryingremoves the water from the material so that enzymatic processes areinhibited. Using freeze-drying is particularly advantageous since thesamples treated thus can be stored and processed at room temperature.

In one embodiment of the invention, the extraction, for example theextraction of the freeze-dried or frozen samples, is aided by furthersteps, e.g. by homogenization and dispersion techniques (Fiehn, Anal.Chem. 2000 and Nature Biotechnology 2000, Sauter, 1991, Roessner, 2000,Bligh and Dyer, 1959, Strand, 1999 and the like).

Thus, the material can be disrupted by high temperatures, vibrating millor other grinding methods, pressure or rapid successive pressurechanges, ultrasonic, shock-wave, microwave and/or Ultraturrax extractionsteps and the constituents extracted better. An extraction method whichallows the process to be automated is advantageous. Thus, for example,an ASE (accelerated solvent extraction), a PSE (pressurized solventextraction), a PFE (pressurized fluid extraction) or a PLE (pressurizedliquid extraction) can be carried out particularly advantageously, wherethe solvent or solvent mixture is forced through the material underpressure and, if appropriate, at elevated temperature (see Björklund,Trends in Analytical Chemistry 2000, 19 (7), 434-445, Richter, AmericanLaboratory 1995, 27, 24-28, Ezzell, LC-GC 1995, 13 (5), 390-398).According to the invention, the extraction is carried out in such a waythat the temperature and the pressure are adapted such that theconstituents are not decomposed and thus the extraction efficacy isgood, for example at a temperature of 0° C. to 200° C., with 20° C.being advantageous or 40° C. to 150° C. being more preferred, 120° C. orless are more preferred. Preferably, the process is carried out at 40bar to 200 bar, more preferably at 70 bar, even more preferably at 100bar to 170 bar. Thus, especially preferred conditions are a temperatureof from 60° C. to 80° C., in particular 70° C., and from 110 bar to 150bar, in particular 140 bar. The extraction time can be between 30 s and20 min, with 10 min being preferred and 5 min or less being morepreferred. Especially preferred is the use of a temperature of from 60°C. to 80° C. and a pressure of from 110 to 150 bar at an extraction timeof less than 5 min. Thus, in accordance with the invention, theextraction conditions are milder than described in the prior art andlead to higher yields and a higher stability of the constituents whichare isolated.

In accordance with the invention, work-up of the extracts can beinterrupted at any point of the process described herein between theabovementioned steps as long as the extracts are stored or preservedstably, for example by freezing them at low temperatures and/orfreeze-drying them. However, it is preferred to avoid interrupting thework-up prior to analysis.

After the extraction, the phases can be divided into various aliquotsand, if appropriate, evaporated, for example to remove volatile acidsand water and/or to prepare the samples for the following process steps,for example with an IR Dancer (shake device under reduced pressure,heated by infrared radiation), a vacuum centrifuge, or by freeze-drying.Evaporation should be carried out under mild conditions, preferably from10° C. to 80° C., more preferably from 20° C. to 40° C., preferablyunder reduced pressure, for example from 100 mbar to 10 mbar, preferablyat 10 mbar, depending on the solvent or solvent mixture. When usingdichloromethane/methanol and/or methanol/water mixtures, it isespecially preferred to reduce the pressure stepwise down to 10 mbar.

The process according to the invention also encompasses steps forseparation and analysis, it being possible for the phases to beseparated in each case via LC, GC and/or CE (capillary electrophoresis).After GC, the constituents can be detected for example by EI-MS(electron-impact ionization and analysis by mass spectrometer) or CI-MS(chemical ionization and analysis by means of mass spectrometer),sector-field mass spectrometer, quadrupole mass spectrometry,time-of-flight mass spectrometry, ion-trap mass spectrometry or Fouriertransform ion cyclotron resonance mass spectrometry, FID (flameionization detector) or Fourier transform infrared spectroscopy, andafter LC for example by means of sector-field mass spectrometry,quadrupole mass spectrometry, time-of-flight mass spectrometry, ion-trapmass spectrometry or Fourier transform ion cyclotron resonance massspectrometry, UV/Vis absorption detection, fluorescence detection, NMRor infrared spectroscopy. The process according to the inventionpreferably comprises an MS (mass spectrometry) analysis, an LC/MS(liquid chromatography coupled to any mass spectrometric detection)analysis, GC/MS (gas chromatography coupled to any mass spectrometricdetection) analysis and/or LC/MS/MS analysis (liquid chromatographycoupled to any tandem mass spectrometric detection), most preferably anLC/MS analysis, CC/MS analysis and/or LC/MS/MS analysis.

After the abovementioned evaporation, the extracts prepared inaccordance with the invention can be taken up in HPLC solvents or HPLCsolvent mixtures and then analyzed by LC. Suitable mobile phases aremixtures of, for example, methanol, acetonitrile or ethanol and/ortert-butyl methyl ether, tetrahydrofuran, isopropanol or acetone and/orwater and/or a salt such as, for example, ammonium formate solution,ammonium carbonate solution, ammonium chloride solution, ammoniumacetate solution, or ammonium hydrogen carbonate solution, or an acidsuch as formic acid, acetic acid, trifluoroacetic acid,pentafluoropropanoic acid, heptafluorobutanoic acid, nonafluoropentanoicacid, undecafluorohexanoic acid, tridecafluoroheptanoic acid orpentadecafluorooctanoic acid, or a base such as, for example,triethylamine, pyridine or ammonia, depending on whether polar orunpolar extracts from step (a) or (b) are to be separated. As a rule, agradient elution is carried out, preferably followed bymass-spectrometric detection, for example MS or MS/MS detection (simpleor tandem mass-spectrometric detection).

To carry out the GC analysis, a transesterification/esterification, inparticular with methanol or ethanol, followed by an oxime formation,preferably a methoximation, can be carried out with the unpolar phaseand/or the polar phase, as described below. Preferably, standardsubstances, for example chromatography and/or internal standards, can beadded to each sample or to the respective extract, for example asolution of odd-numbered, straight-chain fatty acids or hydrocarbons.The extracts are subsequently trialkylsilylated, oximation and/ortrialkylsilylation of the unpolar phase being optional.

These steps can be carried out as described herein, but can also becombined individually with other steps, for example other separation andanalytical methods, as described above, and adapted to suit thesemethods.

In one embodiment in the process according to the invention, anesterification/transesterification is thus carried out in the polarand/or the unpolar phase of the extraction. Theesterification/transesterification is preferably only carried out in theunpolar phase. Preferably, the esterification/transesterificationaccording to the invention of the constituents or some of theconstituents which have been extracted is carried out with anunsaturated or saturated straight-chain, branched-chain or cyclic alkylalcohol having 1 to 8 carbon atoms, for example methanol, ethanol,1-propanol, 2propanol, butanol, pentanol, hexanol and the like. Methanolor ethanol are preferred, methanol being most preferred. The reactiontemperature is preferably between 70 and 150° C., more preferablybetween 90 and 120° C., with 100° C. being most preferred. The reactiontime is preferably between 0.5 h and 4 h, more preferably between 1 hand 3 h. Further solvents which are inert during the reaction may bepresent, for example toluene, dichloromethane, benzene and/orchloroform. Mixtures of the alcohols and/or inert solvents may also beused. However, the solution may also comprise 20% by volume or less ofwater, preferably 10% by volume, with 5% by volume or less being morepreferred.

The amount of other solvents apart from the alcohol mentioned ispreferably 20% by volume to 0% by volume and as low as possible.

In a preferred embodiment, the esterification/transesterification in thepolar and/or unpolar phase is carried out with a volatile acid ascatalyst, preferably with HF, HI, HCl, BF₃, BCl₃, HBr, formic acid,acetic acid, trifluoroacetic acid or trichloroacetic acid, with BF₃,BCl₃ or HCl being more preferred and HCl being most preferred.

A “volatile acid” is understood as meaning an acid which can essentiallybe removed by evaporation, preferably all of the acid can be removed byevaporation.

The use of a volatile, preferably readily volatile, acid such as, forexample, HCl is a further considerable advantage of the processaccording to the invention. Especially preferably, the acid has a lowervapor pressure than the solvent used or the components of the solventmixture or a possible azeotrope of all or some of the componentsincluding the acid itself. In contrast to the process described in theprior art, the use of volatile acids allows the acid residues to beremoved rapidly by evaporation, which can be automated, while in theprior art the acid residues must be removed by washing steps followed bydrying, for example using a desiccant such as sodium sulfate, andfiltration. The process according to the invention is thus considerablybetter suited to high-throughput analysis. The solvent which can beemployed for the esterification/transesterification can be an alkylalcohol with an alkyl radical having 1 to 8 carbon atoms, as describedabove, optionally with a content of an inert solvent or solvent mixture,for example chloroform, dichloromethane, benzene and/or toluene. Amixture of chloroform, methanol, toluene and hydrochloric acid isespecially preferred.

Following the esterification/transesterification, the solvent can beevaporated, preferably to dryness, in order to remove the acid, forexample to remove volatile acids and water and to prepare the samplesfor the following process steps, for example using IR Dancer, vacuumcentrifuge or by freeze-drying. Evaporation should be carried out undermild conditions, preferably at below 80° C., more preferably at below40° C., preferably under reduced pressure, for example at 10 mbar,depending on the solvent or solvent mixture. It is especially preferredto reduce the pressure stepwise to, for example, 10 mbar usingdichloromethane/methanol and/or methanol/water mixtures. The solvent orsolvent mixtures employed can aid the drying step, for example by beingparticularly readily volatile or, as entrainants, by aiding theevaporation of water, as is the case with, for example, toluene.

In a further preferred embodiment, an oxime formation in the unpolarand/or polar phase is carried out in the process according to theinvention. In accordance with the invention, an oxime is understood asmeaning a compound of the structure (I) R-ONR′

where R can be H or an alkyl radical, preferably an alkyl radical having1 to 6 carbon atoms, in particular a methyl, ethyl, propyl, butyl,pentyl or hexyl radical, or a substituted or unsubstituted arylalkylradical, preferably with 5 to 7 carbon atoms in the arylalkyl radicaland with 0 to 2 hetero atoms in the ring or in the chain of thearylalkyl radical, for example a substituted or unsubstituted benzylradical, in particular a halogenated benzyl radical with 1 to 7 halogenradicals, preferably a pentafluorobenzyl radical, it being possible forR′ to be any divalent radical. In accordance with the invention,reactants which can be used for oxime formation are compounds of thestructure (Ib) R—ONH₂, where R is as defined above, preferablyhydroxylamine or O-substituted hydroxylamines or in each case their saltwith a volatile acid, for example hydrochlorides, such asO-alkylhydroxylamine hydrochloride orO-pentafiluorobenzylhydroxylamine-hydrochloride, by processes known tothe skilled worker (see also Fiehn, Anal. Chem. 2000), for exampledissolved in a suitable solvent mixture or solvent, such as, forexample, pyridine. Preferred is a process according to the inventionwherein O-methylhydroxylamine-hydrochloride (II),O-pentafluorobenzylhydroxylamine-hydrochloride (III) orO-ethylhydroxylamine-hydrochloride (IV) is employed for the oximeformation, with O-methylhydroxylamine-hydrochloride (II) being mostpreferred.

The reaction can be carried out for 30 min to 6 h, preferably for 1 h to2 h, preferably at from at least 20° C. to 80° C., more preferably atfrom 50° C. to 60° C. It is especially preferred to carry out thereaction for 1 h to 2 h at from 50° C. to 60° C.

In a further embodiment according to the invention, a trialkylsilylationis carried out in the polar and/or unpolar phase. The trialkylsilylationcan be carried out in accordance with the invention with a compound ofthe formula Si(R^(I-4))₄, R⁴ preferably being anN—C₁₋₄-alkyl-2,2,2-trifluoroacetamide, especially preferably anN-methyl-2,2,2-trifluoroacetamide, as in formula (V). Especiallypreferred is thus the trialkylsilylation with a compound of the formula(V)

where R¹, R² and/or R³ independently of one another can be alkylradicals with in each case 1 to 6 carbon atoms, in particular CH₃, C₂H₅,C₃H₇ or C₄H₉ with the following structural formulae for C₃H₇ and C₄H₉:

R¹ or R² are preferably methyl radicals, R¹ and R² are especiallypreferably methyl radicals. R³ is preferably a straight-chain orbranched alkyl radical having 1 to 4 carbon atoms as described above,especially preferably a methyl radical or tert-butyl radical, with R³more preferably being a methyl radical. A trimethylsilylation ispreferably carried out with MSTFA(N-methyl-N-(trimethylsilyl)-2,2,2-trifluoroacetamide). The reaction canbe carried out for 10 min to 120 min, preferably for 20 min to 60 min,at from 20° C. to 90° C., preferably at between 40° C. and 70° C.

Before the trialkylsilylation, preferably one or more internalstandard(s) and/or chromatographic standard(s) may be added.

In an especially preferred embodiment, the material consists of plantmaterial. Most of the prior art only describes the analysis startingfrom fluids, but not from solid material. The processing of plant cellsdiffers from that of animal cells or tissues in as far as animal cellsonly have a cell membrane, but plant cells are surrounded by a cellwall. For example, plant populations or plant species, for examplegenetically modified or stressed plants, can be extracted. Also,homogenates can be prepared from a large number of organisms. To verifysensitivity, accuracy, precision, variability and reproducibility,standard solutions, or material mixed with standard solutions, can beassayed in the process. To this end, the organic material can besupplemented for example with defined amounts of standard substances.

In a likewise preferred embodiment, the process according to theinvention is optimized for high throughput, low variability and highreproducibility and forms part of a high-throughput analysis.

A process optimized for high-throughput analysis preferably comprisesone or more of the following further steps:

-   (i) harvesting/obtaining the organic material;-   (ii) freeze-drying the material;-   (iii) using extraction mixtures in step (a) and (b) which, when    combined, form one phase, preferably using 80% by volume of methanol    and 20% by volume of water in step (a) and 40% by volume of methanol    and 60% by volume of dichloromethane in step (b);-   (iv) following phase separation and    esterification/transesterification, evaporating the extracts;-   (v) carrying out an esterification/transesterification in the    unpolar phases using a volatile acid, preferably HCl; or-   (vi) analyzing the extracts by MS, LC/MS, LC/MS/MS and/or GC/MS    analysis.

These process steps are optimized for high throughput and the use ofrobots, and manual labor is therefore reduced by at least 10%,preferably 20%, more preferably by more than 30%, most preferably by atleast 50%, over the prior art.

Advantageously, the sample mix-up rate is reduced by more than 10%, morepreferably by more than 20%, even more preferably by more than 30%, mostpreferably by more than 50% by the process according to the inventionand its possibility of using automation and robotics. Likewise, aconsiderably increased reproducibility in a high-throughput analysis isachieved by the process steps according to the invention. The increasedreproducibility of the process according to the invention ischaracterized by an analytical variability which is at least 10%,preferably 20%, more preferably at least 30%, most preferably at least50% less than in the prior art.

The processes described in the prior art are not suitable for ahigh-throughput extraction of metabolites from organic material, or aresuitable to a limited extent only. Known diagnostic extractions relatemainly to analyses of fluids, for example urine, so that these processesare not suitable for working up solid samples, in particular plant cellsamples.

The processes described in the prior art require the freezing andmechanical pulverization of the frozen samples, the separation of theorganic phase from the aqueous phase when preparing total extracts whichencompass both the lipophilic and the polar metabolites, andcomprehensive wash steps of an organic phase with an aqueous solutionfor removing the acid, followed by laborious removal of water from theorganic solvent and, if appropriate, filtration of the sample material;steps which are time-consuming and can only be automated with a highdegree of technical complexity, if at all (Fiehn, Anal. Chem. 2000 andNature Biotechnology 2000). Only the process according to the inventiondiscloses the essential process steps which permit effective andcomprehensive automation in connection with an acceleration of theprocess.

Especially preferred is, accordingly, a process according to theinvention comprising step (iii), more preferably being a processcomprising steps (ii) and (iii), even more preferably (ii), (iii) and(iv), even more preferably (ii), (iii), (iv) and (v). Most preferred isa process comprising all of the steps (i) to (vi). Especially preferredis a process wherein the organic material is extracted by an ASE. Ifappropriate, the extracts can be fractionated further via solid-phaseextraction.

In a further embodiment, the process forms part of a process foranalyzing a metabolic profile, advantageously in the form of ahigh-throughput process, and encompasses the abovementioned processsteps and the following further step: analysis of the resulting data byautomatic peak recognition and peak integration.

Thus, the process according to the invention can be used, for example,to study

-   a) effects of genetic differences on the metabolic profile,-   b) the effect of, for example, environmental conditions, stress,    chemical substances and the like,-   c) the interaction between a) and b) or-   d) the course over time of a), b) or c),

as is the case for example in studies into the effect of one or moresubstances (including, for example, substance libraries) on themetabolic profile of, for example, organisms which are as geneticallyidentical as possible, organisms which are genetically related, down toorganisms which are as genetically different as possible.

The present invention is illustrated by the examples which follow,without this implying any limitation whatsoever.

EXAMPLE 1 Sampling and Sample Storage

Sampling takes place directly in the phytotrone chamber. The plants werecut off using small laboratory scissors, weighed rapidly on a laboratoryscale, transferred into a pre-cooled extraction thimble and placed intoan aluminum rack cooled by liquid nitrogen. If required, the extractionthimbles can be stored in the freezer at −80° C. The time betweencutting off the plant and freezing the plant in liquid nitrogen does notexceed 10-20 sec.

EXAMPLE 2 Freeze-Drying

Care was taken that, during the experiment, the plants either remainedin the sub-zero state (temperatures <−40° C.) or were freed from waterby freeze-drying before they came into the first contact with solvents.

The aluminum rack with the plant samples in the extraction thimbles wasplaced into the pre-cooled (˜40° C.) freeze-drier. The initialtemperature during the main drying step was −35° C., and the pressurewas 0.120 mbar. During drying, the parameters were altered following apressure and temperature program. The final temperature after 12 hourswas +30° C., and the final pressure was at 0.001 to 0.004 mbar. Afterthe vacuum pump and the refrigerating installation had been switchedoff, the system was ventilated with air (dried by means of a dryingtube) or with argon.

EXAMPLE 3 Extraction

Immediately after the freeze drier was ventilated, the extractionthimbles with the freeze-dried plant material were transferred into the5 mL ASE extraction cartridges.

The 24 sample positions of an ASE apparatus (Accelerated SolventExtractor ASE 200 with Solvent Controller and AutoASE software (DIONEX))are filled with plant samples.

The polar substances were extracted with approx. 10 mL methanol/water(80/20, v/v) at T=70° C. and p=140 bar, 5 min heating phase, 1 minstatic extraction. The more lipophilic substances were extracted withapprox. 10 mL of methanol/dichloromethane (40/60, v/v) at T=70° C. andp=140 bar, 5 min heating phase, 1 min static extraction. The two solventmixtures are extracted into the same sample tube (centrifuge tubes, 50mL, with screw top and pierceable septum for the ASE (DIONEX)).

Internal standards were added to the solution: ribitol,L-glycine-2,2-d₂, L-alanine-2,3,3,3-d₄ and α-methylglucopyranoside, andmethyl nonadecanoate, methyl undecanoate, methyl tridecanoate and methylnonacosanoate.

7 mL of water were added to the total extract. The solid residue of theplant sample and the extraction thimble were discarded.

The extract was shaken and then centrifuged for 5 to 10 min at least1400 g in order to accelerate phase separation. 1 mL of the supernatantmethanol/water phase (“polar phase”, colorless) was removed for thesubsequent GC analysis, and 1 mL was removed for the LC analysis. Theremainder of the methanol/water phase was discarded. The organic phasewas again washed with the same volume of water (7 mL) and centrifuged.0.5 mL of the organic phase (“lipid phase”, dark green) was removed forthe subsequent GC analysis, and 0.5 mL was removed for the LC analysis.All of the aliquots removed were evaporated to dryness using theIR-Dancer infrared vacuum evaporator (Hettich). The maximum temperatureduring the evaporation process did not exceed 40° C. The pressure withinthe apparatus was not less than 10 mbar.

EXAMPLE 4 Processing the Lipid Phase for the LC/MS or LC/MS/MS Analysis

The lipid extract which had been evaporated to dryness was taken up ineluent. The HPLC run was carried out with gradient elution.

EXAMPLE 5 Processing the Polar Phase for the LC/MS or LC/MS/MS Analysis

The polar extract which had been evaporated to dryness was taken up ineluent. The HPLC run was carried out with gradient elution.

EXAMPLE 6 Derivatization of the Lipid Phase for the GC/MS Analysis

To carry out the transmethanolysis, a mixture of 140 ml of chloroform,37 ml of hydrochloric acid (37% by weight of HCl in water), 320 ml ofmethanol and 20 ml of toluene was added to the evaporated extract. Thecontainer was sealed and heated for 2 hours at 100° C., with shaking.The solution was subsequently evaporated to dryness. The residue wasdried completely.

Methoximation of the carbonyl groups was effected by reaction withmethoxyamine hydrochloride (5 mg/mL in pyridine, 100 ml) for 1.5 hoursat 60° C. in a sealed vessel. 20 ml of a solution of odd-numbered,straight-chain fatty acids were added as time standards. Finally, aderivatization was carried out with 100 ml ofN-methyl-N-(trimethylsilyl)-2,2,2-trifluoroacetamide (MSTFA) for 30minutes at 60° C., again in a sealed vessel. The end volume prior to GCinjection was 200 ml.

EXAMPLE 7 Derivatization of the polar phase for the GC/MS analysis

The methoximation and trimethylsilylation with MSTFA were carried out asdescribed for the lipid phase.

1. A process for extracting constituents from organic material,comprising (a) extracting with a monophasic mixture of 50% by volume to90% by volume of methanol and 50% by volume to 10% by volume of waterand, optionally, 0% by volume to 40% by volume of a further solvent orsolvent mixture; and (b) extracting with a monophasic unpolar solvent orsolvent mixture, wherein the combined extracts of step (a) and step (b)form one phase.
 2. The process of claim 1, wherein step (a) is carriedout with a mixture of 80% by volume of methanol and 20% by volume ofwater.
 3. The process of claim 1, wherein step (b) is carried out with amixture of 30% by volume to 60% by volume of C₁- to C₆-alkyl alcohol,acetone, acetonitrile, ethyl acetate, dimethyl sulfoxide orN,N-dimethylformamide and 40% by volume to 70% by volume of chloroform,dichloromethane, pentane, hexane, heptane, cyclohexane or carbontetrachloride.
 4. The process of claim 1, wherein step (b) is carriedout with a mixture of 30% by volume to 70% by volume of methanol and 40%by volume to 60% by volume of dichloromethane.
 5. The process of claim1, wherein, in one or more further steps, the constituents arederivatized, chromatographed and/or analyzed.
 6. The process of claim 1,wherein the process comprises at least one of the following furthersteps: i) freezing the material; ii) freeze-drying the material; iii)homogenizing and/or dispersing the material; iv) evaporating an extractor a phase to dryness; iv) carrying out anesterification/transesterification in the unpolar phase; vi) carryingout an oxime formation in the unpolar phase; vii) carrying out an oximeformation in the polar phase; viii) carrying out, in the unpolar phase,a trialkylsilylation where each alkyl radical contains 1 to 6 carbonatoms; or ix) carrying out, in the polar phase, a trialkylsilylationwhere each alkyl radical contains 1 to 6 carbon atoms.
 7. The process ofclaim 1, wherein extracting is carried out at a temperature of from 0°C. to 200° C. and at a pressure of from 40 bar to 200 bar.
 8. Theprocess of claim 6, wherein an esterification/transesterification iscarried out with a volatile acid as catalyst.
 9. The process of claim 1,wherein the solvents or solvent mixtures used for the extractionadditionally comprise up to 5% by weight of buffer salts, acids and/orbases.
 10. The process of claim 1, wherein the phases are analyzed ineach case via LC, GC, MS, LC/MS, GC/MS, and/or LC/MS/MS analysis. 11.The process of claims 1, wherein the material is composed of plantmaterial.
 12. The process of claim 1, wherein the process is part of aprocess for analyzing a metabolic profile and comprises the followingfurther step: analyzing the resulting data by automatic peak recognitionand peak integration.